Site-directed mutagenesis reveals role of mobile arginine residue in lactate dehydrogenase catalysis

The binding of substrates to lactate dehydrogenases induces a marked rearrangement of the protein structure in which a 'loop' of polypeptide (residues 98-110) closes over the active site of the enzyme. In this rearrangement, arginine 109 (a basic residue conserved in all known lactate dehydrogenase sequences and in the homologous malate dehydrogenases) moves 0.8 nm from a position in the solvent to one in the active site where its guanidinium group resides within hydrogen bonding distance of both the reactive carbonyl of pyruvate and imidazole ring of the catalytic histidine 195 (see Fig. 1). Whilst this feature of the enzyme has been commented upon previously, the function of this mobile arginine residue during catalysis has not been tested experimentally. The advent of protein engineering has now enabled us to define the role of this basic residue by substituting it with the neutral glutamine. Transient kinetic and equilibrium studies of the mutant enzyme indicate that arginine 109 enhances the polarization of the pyruvate carbonyl group in the ground state and stabilizes the transition state. The gross active-site structure of the enzyme is not altered by the mutation since an alternative catalytic function of the enzyme (rate of addition of sulphite to NAD+), which does not require hydride transfer, is insensitive to the arginine----glutamine substitution.     

Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.     

Variation in styles of rifting in the Gulf of California

Constraints on the structure of rifted continental margins and the magmatism resulting from such rifting can help refine our understanding of the strength of the lithosphere, the state of the underlying mantle and the transition from rifting to seafloor spreading. An important structural classification of rifts is by width, with narrow rifts thought to form as necking instabilities (where extension rates outpace thermal diffusion) and wide rifts thought to require a mechanism to inhibit localization, such as lower-crustal flow in high heat-flow settings. Observations of the magmatism that results from rifting range from volcanic margins with two to three times the magmatism predicted from melting models to non-volcanic margins with almost no rift or post-rift magmatism. Such variations in magmatic activity are commonly attributed to variations in mantle temperature. Here we describe results from the PESCADOR seismic experiment in the southern Gulf of California and present crustal-scale images across three rift segments. Over short lateral distances, we observe large differences in rifting style and magmatism--from wide rifting with minor synchronous magmatism to narrow rifting in magmatically robust segments. But many of the factors believed to control structural evolution and magmatism during rifting (extension rate, mantle potential temperature and heat flow) tend to vary over larger length scales. We conclude instead that mantle depletion, rather than low mantle temperature, accounts for the observed wide, magma-poor margins, and that mantle fertility and possibly sedimentary insulation, rather than high mantle temperature, account for the observed robust rift and post-rift magmatism.     

Nonsense-mediated decay approaches the clinic

Nonsense-mediated decay (NMD) eliminates mRNAs containing premature termination codons and thus helps limit the synthesis of abnormal proteins. New results uncover a broader role of NMD as a pathway that also affects the expression of wild-type genes and alternative-splice products. Because the mechanisms by which NMD operates have received much attention, we discuss here the emerging awareness of the impact of NMD on the manifestation of human genetic diseases. We explore how an understanding of NMD accounts for phenotypic differences in diseases caused by premature termination codons. Specifically, we consider how the protective function of NMD sometimes benefits heterozygous carriers and, in contrast, sometimes contributes to a clinical picture of protein deficiency by inhibiting expression of partially functional proteins. Potential 'NMD therapeutics' will therefore need to strike a balance between the general physiological benefits of NMD and its detrimental effects in cases of specific genetic mutations.     

Oxidants, oxidative stress and the biology of ageing

Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.     

致第三届中国国际毛纺织会议组织者和参加者

祝贺大会召开 !这次不能如邀访问西安 ,我感到遗憾。祝第三届中国国际毛纺织会议圆满成功。着手研究羊毛及产品的性能与结构之间关系 ,正值此时。大约在 1 92 0到 1 970年这段时期 ,有一批扎实的实验研究项目 ,为研究羊毛结构和机械性能提供了大量资料信息。同时 ,开始了理论模拟研究。这些研究结果请参见由 W.S.Simpson和 G.H.Crawshaw主编、Woodhead Publishing出版社的书 :《Wool:Science and Technology》。尽管这些研究为发展羊毛技术提供了有用的基本信息 ,但是对工业界的直接影响甚小。相比之下 ,羊毛化学的知识为羊毛化学处理…     

Crystallization of RNA

Even as the number of RNA structures determined and under study multiplies, the critical step in X-ray diffraction analysis, growth of single well-ordered crystals, remains at the boundary between art and science. Recent advances in methods of RNA synthesis, purification, and characterization, as well as empirical and technical improvements in crystallization techniques, the development of cryo-crystallography, and the wider availability of bright, tunable, X-rays from synchrotron sources are improving the chances of obtaining RNA crystals suitable for X-ray structural analysis. In this review, we summarize the current status of the design, preparation, purification, and analysis of RNA for crystallization and describe the latest approaches to obtaining diffraction-quality crystals. Received 17 October 2000; revised 6 December 2000; accepted 8 December 2000